Evaluation of LCMSMS Deuterium Scrambling in Clinically Significant Small Molecules

Introduction

Introduction and Objective

LC-MS/MS is a powerful tool that brings numerous benefits to the clinical sample analysis arena. However, due to the complexity of the instrumentation there are some unique challenges that also accompany these benefits. Even following sample extraction and cleanup, matrix effects from the samples can cause interferences or impact ionization efficiency. Deuterium-labeled internal standards are the most common and prevalent labeled internal standards used to compensate for matrix effects. Some deuterium labeled compounds may exhibit hydrogen-deuterium scrambling/exchange in the collision cell which can impact MS/MS transition selection.

In this study we investigated numerous variables that potentially contribute to scrambling in order to ascertain reproducibility and impact on scrambling ratios: influencesof different LC-MS systems (tandem quadrupolevs. quadrupoletime-of-flight), matrix selection, concentration, with and without HPLC, collision energies, and deuterium placement in the internal standard. Numerous small molecules of clinical importance were investigate including: hydroxyvitaminD, testosterone, immunosuppressants, bath salts, and spice cannabinoids.

Serum Extraction:
200μL of sample in serum + 200μL of methanol, vortexed to mix.
Added 1mL of heptane, vortexedfor 30sec,
Centrifuged for 4min at 3000rpm
900μL of top layer dried under nitrogen
Reconstituted in 100μL of ethanol

Comparisons of 25-Hydroxyvitamin D Deuterium Scrambling

25-Hydroxyvitamin D2 25-Hydroxyvitamin D2-d3 25-Hydroxyvitamin D2-d6

25-Hydroxyvitamin D3 25-Hydroxyvitamin D3-d6

Labeled 25-Hydroxyvitamin D2 and D3 Scrambling in Serum

Compound

Label

System

Concentration μg/mL

Transitiondn-1

Transitiondn

Scrambling %dn-1/ dn

25-Hydroxyvitamin D2

d3

Xevo G2

2

398→379

398→380

28.6

0.2

398→379

398→380

35.4

6410

5

416→397

416→398

2.8

416→379

416→380

19.7

398→379

398→380

30.4

50

416→397

416→398

2.8

416→379

416→380

20

398→379

398→380

30.5

d6

6410

5

419→400

419→401

2

419→382

419→383

8.8

401→382

401→383

5.9

50

419→400

419→401

2

419→382

419→383

9

401→382

401→383

5.4

25-Hydroxyvitamin D3

d6

6410

2.5

407→388

407→389

4

407→370

407→371

18.8

389→370

389→371

9.2

Transitions Comparisons for Native and Labeled 25-Hydroxyvitamin D2 and D3 in EtOH on 6410

Parent →Water loss

Compound

Label

Concentration μg/mL

Transition dn-1

Transition dn

Scrambling % dn-1/dn

25-Hydroxyvitamin D2

d3

100

416→397

416→398

2.9

d6

100

419→400

419→401

2

native

50

413→394

413→395

0.5

25-Hydroxyvitamin D3

d6

50

407→388

407→389

4

native

100

401→382

401→383

0.5

Parent →2 Water losses

Compound

Label

Concentration
μg/mL

Transition dn-1

Transition dn

Scrambling % dn-1/ dn

25-Hydroxyvitamin D2

d3

100

416→379

416→380

19.5

d6

100

419→382

419→383

8.9

native

50

413→376

413→377

0.5

25-Hydroxyvitamin D3

d6

50

407→370

407→371

18.9

native

100

401→364

401→365

0.3

Water Loss →2 Water losses

Compound

Label

Concentration
μg/mL

Transition dn-1

Transition dn

Scrambling % dn-1/dn

25-Hydroxyvitamin D2

d3

100

398→379

398→380

30.4

d6

100

401→382

401→383

5.4

native

50

398→376

398→377

0.4

25-Hydroxyvitamin D3

d6

50

389→370

389→371

11.2

native

100

383→364

383→365

0.3

Notes: 25-Hydroxy D2-d6 water loss→2 water loss has same transition as 25-Hydroxyvitamin D3 parent→waterloss. Can be problem if compounds are not well resolved chromatographically.

Conclusions

Scrambling was observed for several of the analytesat select transitions. In all cases, scrambling was mitigated or eliminated by optimizing instrument conditions and transition selection.

Awareness of potential scrambling is important for proper internal standard selection.

Scrambling was observed on both the Agilent 6410 triple quadrupoleand the Waters XevoG2 Q-Tofto approximately the same degree. For a specific transition, scrambling ratios were consistent between solvent and serum.No matrix effects on scrambling.

Scrambling may be mitigated or eliminated by altering instrument conditions and transition selection. However, there is a need to consider potential impact of scrambling on transitions chosen for optimal sensitivity.

Deuterium-labeled internal standards are a viable option for LC-MS/MS analysis with selection of the appropriate transition. Deuteratedstandards can be more cost effective than 13C labeled internal standards, more widely available and with lower cost per test.13C labeled internal standards are most effective when deuterium scrambling issues can not be resolved.